Production of sweet protein, brazzein, as ubiquitin fusion.
Brazzein has the potential to be used extensively as a commercial sweetener because of its high thermostability and water solubility. The drawbacks of this sweet protein are that extraction of brazzein from its natural source is expensive while other developed production methods are not efficient. C...
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| Format: | Final Year Project |
| Language: | English |
| Published: |
2012
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| Online Access: | http://hdl.handle.net/10356/49277 |
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| Institution: | Nanyang Technological University |
| Language: | English |
| Summary: | Brazzein has the potential to be used extensively as a commercial sweetener because of its high thermostability and water solubility. The drawbacks of this sweet protein are that extraction of brazzein from its natural source is expensive while other developed production methods are not efficient. Currently there is no gene for brazzein isolated from its plants of origin; therefore a synthetic gene is designed based on the optimised expression and codon biased of E.coli. The construction of pET30 expression vector helped to increase the expression levels of brazzein in E.coli and MALDI-MS confirmed that the purified protein was the desired brazzein-ubiquitin protein. However, this fusion of brazzein to ubiquitin also rendered the protein to be tasteless, possibly as a result of the inability of brazzein to adopt its native state. By analysing the results, it was affirmed that the C-terminal is important for brazzein to fold into its bioactive conformation to elicit its sweetness. This protocol was also attempted in S.cerevisiae but unable to replicate the same results, possibly because of instability of YEp181 plasmid. Hence, further experiments can be conducted to resolve these issues to produce brazzein in large quantity through yeast biotechnology. |
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